Open access peer-reviewed chapter
Abstract
Liver disease is one of the major chronic disabilities around the world. It is known that global casualties are increasing because of virus C infection, alcohol consumption, or non-alcoholic circumstances. One of the main derived comorbidities of liver disease is the hepatic encephalopathy (HE), a severe neuropsychological syndrome derived from the acute or chronic liver disease. A key feature accounting for HE symptoms in cirrhotic patients is brain edema, which is triggered by hyperammonemia. In basal conditions, ammonia can be metabolized in the central nervous system (CNS) by astrocytes, which synthetize glutamine using ammonia and glutamate as substrates. In hyperammonemic conditions, astrocytes synthetize large amounts of glutamine generating a hyperosmotic condition, inducing these cells to become swollen in shape, invoking the characteristic symptom clinically manifested in patients with HE, as brain edema; this condition is regulated by water channels called aquaporins (AQPs) and by other molecules such as myoinositol. Experimental evidence suggests that some small non-coding RNAs may regulate AQPs expression both in vivo an in vitro and that some pharmacological interventions improve cognitive impairment in cirrhotic patients. It is undeniable that astrocytes and the different signaling pathways beneath its plasma membrane play a crucial role in liver disease-derived HE and represent some of the novel pharmacological targets to treat comorbidities of the liver disease.
Keywords
- liver
- hyperammonemia
- encephalopathy
- astrocytes
- astrogliosis
1. Introduction
1.1. General aspects of liver disease and hepatic encephalopathy
Liver disease is one of the leading non-infectious pathologies affecting people around the world. In its report from 2015, WHO indicates that the advanced form of liver disease, meaning cirrhosis, is among the 20 most frequent (2%) causes of death (~1.62 × 106) around the globe (http://www.who.int/). Hepatic encephalopathy (HE) is a pathological condition that represents the neuropsychiatric disorder derived from liver disease. It is known that about 80% of patients with liver disease (depending on severity), may develop HE, which represents one of the major complications leading to death in about 90% of patients with acute liver failure (ALV) [1]. HE has been classified by the Hepatic Encephalopathy consensus Group, at the World Congress of Gastroenterology in 1998, in type A, associated with ALV; type B related to porto-systemic bypass; and type C related with chronic liver disease, mainly cirrhosis [2]. Further classification includes the severity of symptoms that includes the subcategory called covert (CHE, also known as minimal), persistent (PHE), and overt (OHE). The former is mild in manifestations and is hard to diagnose without specific neurophysiological and neuropsychological tests, such as the visual-based flicker test, the psychometric hepatic encephalopathy score (PHES), the repeatable battery for assessment of neuropsychological status (RBANS), the inhibitory control test (ICT), the cognitive drug research (CDR), and the most recent STROOP App test (
2. Astrocytes as mediators of the pathophysiology of HE
In this and further sections, we will try to introduce to the reader into a review about recent findings that illustrates how the physiological functions of the liver, or to a better extent, a lack of them, may harm at different levels, the metabolic and physiological homeostasis over brain cells and function.
2.1. Ammonium metabolism
The liver is responsible to detoxify the majority of endogenous or exogenous toxic compounds produced by the metabolic or catabolic activity within the organism, including pharmacological metabolites, ammonium, bilirubin, bile salts, etc. Several factors may affect liver functions, such as increased consumption of a fat diet, another one is the chronic alcohol abuse; while the infection by hepatitis C virus (HCV), pharmacological intoxication (mainly paracetamol), complete the pathological scenario. The fatty liver, also known as steatosis (non-alcoholic fatty liver disease or NAFLD), has been called the first hit in liver damage, which when not properly attended, could result in a chronic inflammation or steatohepatitis (NASH). NASH could evolve to fibrosis and eventually to a more critical stage, cirrhosis. Cirrhotic patients suffer a wide spectrum of comorbidities [9]. Among them is hepatic encephalopathy (HE), a debilitating psychological condition where the patients present a variety of symptoms including loss of memory, mild or manifested tremors, and most importantly, increased plasma levels of ammonia. In basal conditions, ureotelic organisms like humans, should contend with the ammonia derived from the gastrointestinal bacterial activity and muscle metabolism, by converting it to carbamoyl-phosphate (CP), a substrate of the urea cycle, CP is then converted to citrulline, the precursor of arginosuccinate; arginosuccinate is then transformed to arginine and by the action of arginase, arginine is converted to urea and ornithine; all of these reactions takes place within the mitochondria and cytosol of the hepatocytes, resulting in the release of urea into the bloodstream and then in urine [10]. However, in pathological conditions like cirrhosis, hepatocytes are unable to produce urea; therefore, ammonium in its form of gas (NH3) easily diffuses to the blood brain barrier (BBB). Once within CNS, ammonium is metabolized primarily by the astrocytes, a group of specialized glial cells capable of modulate inhibitory and excitatory neurotransmission. In its form of ion (NH4+), ammonia reaches the astrocytes where a different transporter mediates its translocation throughout the plasma membrane. Among this transporters are the Na+/K+-ATPase pump, the Na+/H+-ATPase antiporter and Na+/K+/2Cl−, K+/Cl− symporters [5]. Astrocytes metabolize the ammonium by the so-called glutamate-glutamine cycle (GGC). The GGC produces glutamine (Gln) through the action of glutamine synthetase (GS), delivering it to neighboring glutamatergic neurons (Figure 1). In neurons, glutamine is subject to the opposite reaction, mediated by the enzyme glutaminase, whose products are glutamate and ammonium. Glutamate is packaged into synaptic vesicles and released to the synaptic cleft in response to electrochemical stimulus, while ammonium is released to the extracellular space [11]. Once released, glutamate binds to ionotropic (N-methyl-d-aspartate (NMDA) or AMPA) receptors, or metabotropic (mGluR1 and mGluR2) receptors in the postsynaptic neuron and astrocytes [11]. Unbound released glutamate can be recycled when captured by the excitatory amino acid transporters (EAAT) located in the astrocytes membrane. These events represent the final step in the GGC.
Figure 1.
The glutamine (Gln)-glutamate (Glu) cycle within astrocytes. Glutamine synthase (GS) mediates the reaction of ammonia (NH4+) and Glu to produce Gln. Gln is then exported by a specific transporter (illustrated here as a small black cylinder) to the glutamatergic neurons near the astrocyte. The cycle completes when Glu is released to the synaptic cleft and then binds to NMDA receptors or is recaptured by astrocytes to began the cycle. See text for details.
On the other hand, besides GGC, astrocytes may use other systems to titrate ammonium from bloodstream. Those based in the use of the branched chain amino acids (BCAA) on one side, or the use of L-ornithine and L-aspartate in the other. In the first case, BCAA are metabolized by the enzyme branched-chain amino acid transaminase (BCAT) particularly enriched in the cytoplasm and axons of glutamatergic and GABAergic neurons [12]; cytoplasmic BCAT enzymatically mediates the synthesis of branched-chain ketoacids (BCKA), which in turn generates both, a precursor for the tri-carboxylic acid cycle (TCA) in the form of Acetyl-CoA and the excitatory amino acid glutamate; glutamate in turn, can be metabolized by means of the GS to produce glutamine; glutamine is deaminated by the phosphate-activated glutaminase (PAG) which is also present in the cytoplasm of neuronal and glial cells [13]. The second one, the L-Ornithine and L-Aspartate system, operates to generate glutamate by means of the ornithine- or aspartate-aminotransferase (OAT, AAT); both enzymes are enriched in the skeletal muscle, thus contributing to stimulate the glutamine production in this tissue by the GS, diminishing the ammonia derived from circulation [5]. L-ornithine and L-aspartate represent one of the most frequent therapeutic approaches to improve ammonia levels in cirrhotic patients, as we shall see later.
2.2. Astrocytes and the effect of hyperammonemic conditions within CNS
It is known that NH4+ and K+ have comparable physical and chemical properties, so that any change or increase in NH4+ concentrations may activate the Na+ transport by the Na+/K+-ATPase pump, leading to abnormal neurotransmission in neurons or even astrocytes [14]. NH4+ enters to the astrocytes and induces the increase in the activity of the GS; this event results in the accumulation of glutamine. As intracellular glutamine concentrations rise, the osmotic pressure does it in parallel, provoking the astrocytes to become swelling and to activate two main mechanisms to counteract this morphological change [15]. The first one consists of the release of an osmotic regulator, myoinositol; myoinositol leaves the astrocyte to redress the osmotic balance within the cell to prevent astrocyte and cerebral edema; while the other one is to increase the activity of the water channels called aquaporins (AQPs). One of the main water channel transporters related with regulation of the osmotic response in astrocytes, is the aquaporin 4 (AQP4) [16]. It has been reported that AQP4 increases its activity under hyperammonemic conditions, allowing the H2O molecules to enter into the cell, thus relieving the osmotic pressure induced by the accumulation of glutamine [17]. Until then, it was not clear whether the increased activity of AQP4 was the result of a higher transcriptional activity or an increased rate of mRNA translation [18]. Evidence in favor of both options had been reported. In two separate reports, Jalan et al. and Norenberg et al. demonstrated independently the increased expression of AQP4 protein, in a chronic or acute
As we have seen, hepatic failure is closely related to astrocyte low grade and brain edema mediated by water movement throughout the AQP4 channel. Among the pathogenic factors contributing to aggravate this condition are the free radical production, the MAP-kinase activity, and the induction of the mitochondrial permeability transition (MPT) process. MPT is well known to occur in response to the sudden intra-cytoplasmic increase of molecules around 1.5 kDa in size that enters the mitochondria mediated by the so-called permeability transition pore (PTP), which is located at the inner membrane [24]. These phenomena critically compromises mitochondria homeostasis and the metabolic function of many crucial processes like the TCA and ATP synthesis, promoting the generation of reactive oxygen species (ROS). It seems that HE promotes the generation of ROS, therefore inducing the MPT due to the oxidative stress, because indirect evidence demonstrates that antioxidants are able to reduce MPT. Norenberg et al. demonstrated that MPT is involved in the ammonium-induced astrocyte swelling after their
2.3. Systemic and local inflammatory pathway effects on astrocyte disturbances
Another important mechanism of regulation of astrocyte physiology in response to hepatic failure is the activation of the pro-inflammatory response. In recent years, an increasing role of the inflammatory-related pathways in response to ammonia-induced brain dysfunction has been reported. The cytokines promoting brain damage are TNFα, interlukin-6 (IL-6), interlukin1-beta (IL-1β), IL-18, and many others. However, there is controversy about the role between these pro-inflammatory cytokines in both acute and chronic hepatic failure. Several groups have provided evidence of a positive correlation in this interaction in human subjects or animal models. In 2007, Wright and colleagues determined the TNFα, IL-6, and IL-1β artery blood levels and found a positive correlation of these cytokines with intracranial pressure in ALF-affected humans [26]. Further evidence indicates that the same cytokines are elevated, at least at the transcriptional level, in the brains of a rat ALF model [27]. A recent interesting proposal about the role of these circulating inflammatory interleukins indicates that they might interact with certain blood-brain barrier (BBB) endothelial cells to reach the CNS, acting synergistically with ammonia. This synergistic model has been expanded through the identification of additional factors such as GABA produced by certain bacterial families within the gut, contributing to modify the GABAergic tone that reach the CNS via the vagal afferent pathway [28]. In view of these facts, systemic inflammation is in part responsible for the astrocyte and neuronal network alterations in both acute and chronic liver disease. A major player in the systemic inflammation effects over CNS in these pathologies is a member of the danger-associated molecular patterns (DAMPS) called high mobility group box protein 1 (HMGB1), which in basal conditions regulates transcriptional activity [29]; however, in pathological conditions, it can be released passively from damaged cells into the extracellular space in response to pro-inflammatory stimuli, such as LPS; HMGB1 can also be actively released by a JAK/STAT acetylation-mediated process, from immunocompetent cells [30]. In a recent study, Ohnishi et al. showed that HMGB1 significantly decrease the expression of AQP4 mRNA and protein in cultured astrocytes; conversely, the intra-cerebroventricular injection of HMGB1 in adult rats, slightly increase AQP4 protein expression and as expected, the brain edema; further, exposure of microglia to a HMGB had a significant effect over IL-1β mRNA expression and protein release, which apparently regulates AQP4 increased expression; the authors suggest that the pathway IL-1β-NF-κB-AQP4 in microglia might regulate the brain edema in response to HMGB1-mediated systemic inflammation [31]. Whether this pathway might occur with other pro-inflammatory cytokines such as TNFα, IL-6, or IFNγ, remains to be investigated.
The unusual extracellular histone proteins represent another piece in the puzzle of the systemic inflammation response. It has been showed that in ALF or HBV infected patients, increased concentrations of circulating histones correlates with an immunostimulatory effect, leading to multiple organ injuries [32, 33, 34].
Along with systemic and local brain inflammation, the increased ammonia and manganese levels in the brain also alter the TCA cycle, promoting lactate accumulation and the promotion of dopaminergic cell death, as well as the generation of reactive oxygen species (ROS) in basal ganglia, and contributing in this way to the parkinsonism-like behavior and cognitive impairment observed in cirrhotic patients [36]. In this line of evidence, oxidative stress is thought to be of relevance for ammonia toxicity in HE. Molecular studies indicate that acute ammonia loading, mechanical or drugs stressors like hypo-osmolarity, diazepam, and TNFα,
2.4. The role of the intestinal microbiota in the gut-liver-brain axis
The intestinal microbiota has evolved as a new and relevant player in the pathogenesis of several intestinal and non-intestinal diseases. As the liver is the organ in closest contact with the intestinal tract, it is potentially exposed to bacterial components and metabolites. In physiological conditions, nutrients and bacterial compounds translocate to the liver via the portal circulation and contribute to the host homeostasis. As the epithelial wall and mucus layer act as physical barrier to impede that the most of the bacterial components or even bacteria reach the blood flow, it is usually accepted that a small quantity of these compounds enter the portal venous flow, and a tight balance on the immune response is achieved in order to fight against potential exogenous insults. However, in pathological conditions, such a physical barrier can become more permeable to bacterial components, specially lipopolysaccharide (LPS), flagellin, peptidoglycan, and microbial nucleic acids [40]. Evidence about the effect of gut microbiota over hepatic physiology has been demonstrated. In 2010, Gupta and co-workers found that small intestine bacterial overgrowth (SIBO) was a hallmark in cirrhotic patients suffering of HE [41]; in the same year, Jun et al. published evidence about the direct association of SIBO with the peripheral founding of bacterial DNA in cirrhotic patients [42]; later on, in 2012, Henao-Mejia et al. demonstrated that genetic deficiency of inflammasome, a protein complex considered the sensor of endogenous and exogenous pathogen-associated molecular patterns (PAMPS), induced the accumulation of bacterial components in the portal circulation, with the consequent liver damage effect [43]. One important fact about the bacterial overgrowth in cirrhotic patients is that the translocation of bacteria or any of its components promotes and aggravates the liver illness. There are several reports indicating the differences in the fecal microbiota of various populations around the globe, specifically in patients with liver cirrhosis in comparison with healthy subjects. In general, these studies coincides in founding that the
Another significant consequence of SIBO is the higher tendency of the microbial community to increase the synthesis and release of organic compounds with a metabolic effect over a wide number of host cells. For example, it has been demonstrated that ingestion of
Here, we can say that the net result of the so-called “
2.5. Genetic and epigenetic effects induced by HE in astrocytes
Epigenetic modifications are heritable and reversible stable marks that do not modify gene sequences
On the other hand, we know that non-coding RNAs (ncRNA) are important players in epigenetic mechanisms regulating cell fate and metabolism. Among the ncRNAs with relevant physiological roles are small RNAs and the long non-coding-RNAs (lncRNAs). A variant of small RNAs includes the microRNAS (miRNAs), which are beginning to acquire significant roles in coordinating astrocyte gene expression. miRNAs are small RNAs (~22 nt in length) that controls gene expression through their binding to the 3′-untranslated region (UTR) in mRNAs, leading transcripts to degradation or translational repression [59]. Long noncoding RNAs (lncRNA) on the other side are sequences of about 200 nt in length, which regulate the expression of neighbor protein-coding-genes in a phenomenon called “transvection” (for a systematic review see [60]). In the last decade, there has been an increase in the number of reports demonstrating the role of both miRNAs and lncRNAs in the physiopathology of HE. Two of the first reports regarding the role of two specific miRNA in the regulation of astrocyte cell swelling came from Singapore; in 2010 and 2012, Jeyaseelan and colleagues published in separate papers that miR-130a and miR-320a directly affect the AQP4 expression in the CNS. In the first case, the role of miR-130a is a non-canonical regulation, because the repression mechanism occurs at the transcriptional level over the AQP4 M1 gene promoter [61], while the mir320a was described as a modulator of AQP1 and AQP4 in a model of cerebral ischemia [62]. In a more wide recent study, Häussinger and co-workers explored the global miRNA profile using NH4Cl stimulated rat brain astrocytes
Figure 2.
The expression of miR-29 and miR-148 diminished in response to hyperammonemia in vivo and in vitro. Total RNA extracted from the whole brain of bile duct ligated (BDL) mice or control mice (Sham), as well as primary astrocytes (CD1) or the astrocytoma cell line T-98 in the presence or absence of NH4Cl, was used to evaluate the expression by means of qPCR. The data represent the mean expression of at least three replicates in each case (unpublished data).
The lncRNAs, on the other hand, had been more less studied in the context of liver-associated CNS pathologies; however, in a recent study in which 35,923 lncRNAs were screened using microarrays, Silva and colleagues found that 380 and 486 transcripts were upregulated or downregulated in a mouse model of acute liver failure. The authors found that some of these lncRNAs might be related to the cytokine-receptor interaction, MAPK, insulin, NFκB, and TNFα signaling pathways, all of them related to the inflammatory response; the authors also found that the lncRNA uc007pjf.1 associated with a guanine nucleotide exchange factor (NET1), which regulates RhoA, is involved in the cytoskeleton dynamics, suggesting a relationship of this factor with the astrocyte cell swelling observed in HE [64]; these data are of relevance because contributes to corroborate the role of some genetic pathways already related with ALF and certainly established the roots to new discoveries; however, these findings are the result of a combination of cellular phenotypes within CNS and are hardly extrapolated solely to astrocytes, neurons, microglia or even to non-differentiated precursor cells residing in the frontal cortex; complementary
2.6. Clinical data in osmoregulation and systemic infections in cirrhotic patients with HE
The maintenance of a constant cell volume is a critical problem of all cells. Cell swelling or shrinkage is undesirable for normal cellular function. Changes in the cell volume mostly occur due to changes in the extra or intracellular osmolarity. As most cell membranes are freely permeable to water and do not possess water pumps in their membranes, cells will shrink or swell in response to changes in the tonicity of the extracellular fluid (ECF) [65]. Cells will shrink in a hypertonic ECF, while they swell in a hypotonic ECF. Similarly, when the tonicity of the intracellular space of the cells increases, the cells try to compensate it and increase the osmolarity by the uptake of water from the ECF—and consequently swell. Extra- and intracellular osmolarity is determined by the concentrations of several compounds, such as ions, amino acids, etc. Consistent with the adaption against the rise of intracellular osmolarity in patients affected by CLF, it has been proposed that reductions in brain concentrations of myoinositol are implicated in the pathogenesis of ammonia-induced brain edema [66].
In the same line of evidence, it is difficult to elucidate whether the release of taurine (an atypical aminoacid with osmoregulatory properties) from the astrocytes to the ECF is an osmoregulatory response to increased intracellular glutamine and/or cell swelling, since other mechanisms that increased intracellular osmolarity may lead to the cellular release of taurine and taurine may also be released from neurons. Taurine has been related with reversal of hepatic hypertension in a rat model of liver cirrhosis; in a study from 2009, Liang and colleagues found that natural taurine significantly decreased the portal venous pressure, resistance and flow, and markedly decrease the nitric oxide (NO) and cyclic guanine-monophosphate (cGMP), with a concomitant reduction in the pathological status of liver tissue damage and the expression of collagen 1 (COL-1), COL-III, and transforming growth factor-β1 (TGF-β1) [67]. Taurine also have clinical uses in the form of a hydrophilic bile acid called tauroursodeoxycholate (TUDCA) for treatment of primary biliary cirrhosis (PBC). In a randomized cross-over study with 12 female patients suffering from PBC, the use of TUDCA showed to improve the enrichment of biliary ursodeoxycholate (UDCA) and was better absorbed than ursodeoxycholate and undergoes less biotransformation than UDCA, thus suggesting that TUDCA is clinically relevant for the treatment of cholestatic liver diseases [68]. As taurine is synthetized in both liver and brain it is plausible that HE might impact on taurine metabolism and biological action. Several reports have indeed described the pathological disturbances in patients with chronic liver disease as well as in liver failure murine models. In both cases, the taurine concentrations were significantly diminished [69, 70]. Astrocytes synthetize and stored taurine, which is involved in ion movement across CNS, mainly K+ and Ca2+; therefore, is not rare to consider that neural excitability modifications in cirrhotic patients occurs. Evidence in favor of this hypothesis was reported in an experimental model of ALF, in which CSF taurine concentrations significantly increased in a positive correlation with early progression of HE [71]. As taurine has osmoregulatory actions any increase or decrease in its extracellular concentrations could have an impact in the pathogenesis of acute liver failure as has been demonstrated [72]. In contrast, taurine concentrations in chronic liver failure are not changed, but the release of taurine into the extracellular space could represent a control mechanism for volume regulation of astrocytes [73, 74].
Another important metabolite that has been related with HE is the acidic calcium binding protein S100β, produced by astrocytes in the CNS and apparently secreted in response to oxidative stress, exerting its paracrine or autocrine effects on glia, neurons, and microglia [75, 76, 77]. S100β is a multifunctional protein that can be measured in basal conditions in CSF and is barely detected in circulating blood flow; however, in pathological conditions, its serum levels might increase, indicating neuronal or astrocytic damage. Our group and others have begun to investigate the diagnostic efficacy of S100β as a biomarker to detect low grade HE in cirrhotic patients. Recent findings from our laboratory indicate that cirrhotic subjects with OHE have higher serum levels of S100β when compared with non-HE cirrhotic or cirrhotic patients with MHE or control subjects, and that 0.13 ng/mL of S100β is the best cut-off for the diagnosis of HE (83 and 64% of sensitivity and specificity, respectively) [78]. These results are in accordance with previous data in which S100β and neuron-specific enolase (NSE) were evaluated in a small Egyptian cohort of 52 subjects, where 62 and 38% of 29 cirrhotic patients had HE grade 1 or 2, respectively; these groups were compared with non-HE cirrhotic or healthy subjects. Here, NSE showed non-significant differences among the groups, but S100β was increased in serum samples from grade 1 and 2 HE, compared with cirrhotic or healthy subjects; in addition, a significant positive correlation was found between S100β levels and plasma ammonia, in all patients; the main conclusion in this report was that S100β serum levels could be a useful surrogate marker with more than 90 and 50% of specificity and sensitivity, respectively, for detection of mild cognitive impairment in cirrhotic patients, before they progress to more advanced stages of HE [79]. In a similar study, Wiltfang et al. reported that a cut-off value of 112 pg./mL or 0.11 ng/mL of serum S100β, practically the same value found in our group of patients, is able to predict subclinical porto-systemic encephalopathy with a 100 and 57% of specificity and sensitivity, respectively [80].
Osmotic abnormalities account for the cognitive impairment in cirrhotic patients and some clinicians are using the magnetic resonance (MR) in order to clearly demonstrate a relationship among water, manganese, glutamate, glutamine, and myoinositol levels and the degree of brain damage in patients with liver disease. MR might be useful to diagnose brain abnormalities elicited by hyperammonemia, especially in specific regions like the
Figure 3.
Cirrhotic infected patients have significant abnormalities in the glutamine/glutamate (Glx) ratio and NAA brain levels. See text for details.
Besides those data indicating that brain metabolites are also implicated in the pathophysiology of HE, another important issue in end-stage liver disease is the local blood flow. Cirrhotic patients with portal hypertension suffering ascites, hepatorenal syndrome and HE, usually also had distorted cerebral blood flow (CBF), which severely impact their skeletal muscle, brain, and kidney irrigation as a result of vascular resistance. It has been a long way since the 1960s decade, when one of the first studies about cerebral hemodynamics in cirrhosis came into light [82]. Since then, a plenty of studies have contributed to understand the role of vascular pressure over the pathology associated with HE. In 1969, Bianchi-Porro and co-workers reported that after portacaval shunt surgery, cirrhotic patients, had a significance increase in CBF and a significant decrease in cerebral vascular resistance (a measure of CBF and metabolism) and the metabolic rate of glucose and the glucose:oxygen quotient did also increase in these patients; the authors suggested that the increased CBF was related to metabolic problems, and that this increase accelerates the removal of toxic substances and allows the disposal of brain metabolites capable of neutralize toxic compounds [83]. It is now well accepted that cirrhotic patients have decreased CBF, portal hypertension, and splanchnic vasodilatation, associated with a hyperdynamic circulatory state, all of which can affect CBF. In a more recent study, we described a maneuver in which we used the transcranial Doppler (TCD) technique (transmission of ultrasound beam through the skull using a pulsed Doppler sectorial probe with a 2 MHz transducer), to measure in an non-invasive and reliable way, the cerebral autoregulation, meaning the physiological mechanisms that maintain CBF at an appropriate levels in response to vasoconstrictors or vasodilators and the middle cerebral artery (MCA) velocities. From here, two functional cerebral hemodynamic indexes were evaluated, (a) the pulsatility index (PI) which assesses the arteriolar vascular integrity and (b) the breath holding index (BHI), which measures cerebrovascular reactivity (CVR). We compared the TCD measures of cirrhotic non HE (n = 30), cirrhotic HE (n = 30) and healthy subjects (n = 30). Here, we found that major basal vessel integrity was not compromised because there were no differences among the three groups when the left and right cerebral arteries were evaluated. However, we observed significant differences in PI and BHI between cirrhotic and control subjects and when the compensated status was taken into account, the PI and BHI was significantly increased and decreased, respectively, when decompensated (CTP > 7 and MELD score ≥ 14) patients were compared with compensated and healthy groups. Similarly and more importantly, when HE status was included in the analysis, the results were the same as before, which means that disturbances in CBF is a novel pathophysiological pathway in HE, and opens an new way for treatment and prevention. Patients with decompensated cirrhosis or HE have a higher risk of cerebral hypoperfusion related with microvascular damage and the ability for autoregulation, which is and important feature for systemic blood pressure sudden changes, particularly hypotension, which can promotes HE [84]. These findings were similar to those reported by two separated groups, one located in Turkey, were 50 decompensated group of cirrhotic patients was compared with 50 healthy volunteers using the same TCD approach [85]; while other in China recruited a healthy control group (n = 40), a cirrhotic without HE group (n = 52), a cirrhotic with MHE group (n = 21) and a cirrhotic with OHE group (n = 19) [86]. Here, in the first case, the authors analyzed the spectra signal derived from the systolic velocity, diastolic velocity, mean flow velocity (MFV in cm/s), pulsatility, and the resistive indexes (PI = Peak systolic velocity (V
3. Novel pharmacological findings over HE treatment
Despite being one of the most frequent and best studied associated pathologies of liver dysfunction, HE still represents a bigger challenge to clinicians due to its devastating effects over cognitive functions. Still, there is no clear consensus about its appropriate treatment because of its complex physiology involving inflammatory responses, neurosteroid-like compounds, reactive oxygen species, etc. In addition, there is another layer of complexity represented by the co-infections and organ failure that many patients develop in the course of the pathology. Nowadays, there had been a lot of attempts to circumvent the signs and symptoms of HE, both experimentally and clinically. One example of this is the use of one of the most popular anti-inflammatory drugs used to treat cirrhotic patients, infliximab, which reduces peripheral inflammation, directly impacts over neuroinflammation, and restores the altered neurotransmission and cognitive impairment, as well as the reversal of activation of microglia and astrocyte GABA transporters (GAT1, GAT3). Infliximab also reduces the synthesis and release of pro-inflammatory cytokines, such as TNFα, IL-1β, etc., as demonstrated by Dadsetan et al. in a murine model of HE [87, 88]. These data are still scarce in human subjects, although some advance have been reported. In an initial trial, Sharma and co-workers reported that patients with severe alcohol-associated hepatitis, who received a single dose of infliximab (5 mg/kg IV), have an improvement of the Maddrey’s discriminant factor (DF)—useful in the prediction of short-term prognosis—serum TNFα, C reactive protein (CRP), MELD score and total neutrophil count compared with the before-treatment parameters; interestingly, among the patients who survived only 8% had HE at admission, while among those who died, 67% suffer from HE; the authors concluded that HE at admission of the trial, Lille score and delta bilirubin, predicted 2-month mortality and that infliximab should be carefully used to treat alcoholic hepatitis [89]. Besides the use of infliximab, other therapies aimed to reduce the production of ammonia by the intestinal microbiota, especially the coliforms, have been widely reported. Treatment with lactulose (a non-absorbable disaccharide) or non-absorbable antibiotic rifaximin, are two of the most frequent therapies for cirrhotic patients suffering from HE. The metabolic activity of colonic bacteria which produce short-chain organic acids when metabolize lactulose lowering the pH of the gastrointestinal tract, is the main effect of the disaccharide for the removal of nitrogen; the acidic environment also results in the change of ammonia to ammonium (NH4+) a non-absorbable form of nitrogen which diminishes the circulating ammonia. Further mechanisms include the laxative effect for removal of nitrogen-containing compounds from the gut. In a concise Cochrane review from last year, 38 clinical trials were evaluated and their findings indicate that non-absorbable disaccharides have beneficial effects when compared with placebo/no intervention on mortality, HE, liver failure, hepatorenal syndrome and variceal bleeding; secondary outcomes such as quality of life, also were favored when non-absorbable disaccharides were administered [90]. Additionally, experimental data indicates that lactulose promotes neuro- and astrogenesis in the hippocampus of rats with HE as well as the reduction of plasma ammonia, the locomotor activity impairment and neuronal hyperactivity in brain areas related with locomotor activity [91]. These data are in accordance with those data indicating that Lactulose do also improved cognitive function in MHE patients, as reviewed by Luo et al. [92]. The non-absorbable antibiotic, rifaximin, is the most common additive therapy along with lactulose to treat cirrhotic patients with HE. In a recent prospective observational study, 60 HE patients were divided into two groups, one receiving rifaximin alone and the other one received rifaximin plus lactulose for 7–15 days until discharge from hospital or death. In this study, the authors found that both groups were effectively improved in their mental scores, although they conclude that the combination of both substances was effective, but not superior to lactulose alone in the treatment of HE [93]. The resolution of overt HE has also been tested using rifaximin in comparison with other antibiotics. A meta-analysis of these trials indicates that rifaximin treatment was more likely to resolve an episode in patients with OHE besides an improvement in secondary prevention of HE [94]. Other treatment regimes devoted to reduce the serum ammonia levels, such as polyethylene glycol (PEG), sodium benzoate, odium phenylacetate, glycerol-phenylbutyrate (GPB), ornithine-phenylacetate (OP), phenyl-acetyl-glutamine (PAGN), and a carbon microsphere adsorbent (AST-120) among others, had been also tested for nitrogen removal (for a concise review see [95]).
On the other hand, it is more common to see the rise of many biological compounds that previously were used to treat distinct pathologies; an example of this is Artesunate, a water-soluble hemisuccinate derivative of the Chinese herb
Although the mechanisms of astrocyte cell swelling are still controversial, some authors propose that the plasma membrane depolarization plays a crucial role, while others suggest that the ionic homeostasis is equally important [103, 104]. In addition, glutamine, manganese, pH changes, and the neurosteroids are also implicated in this process. However, both,
A most recent study, conducted
L-ornithine and L-aspartate (LOLA) are urea cycle substrates that can lower ammonia levels in cirrhotic patients. The first is a substrate of the ornithine-aminotransferase (OAT) enzyme, which converts ornithine into glutamate or glutamate-semialdehyde, while L-aspartate is converted by the aspartate-aminotransferase into glutamate or oxaloacetate [5]. A complete review about the effect of LOLA in cirrhotic patients has been published and one of the main conclusions was that LOLA is effective in patients with OHE and less beneficial for those having MHE and had no effects on patients with acute liver failure [111, 112].
An intriguing method to treat liver-affected patients is based in Ayurveda. According to Wikipedia, Ayurveda is a system of medicine with roots in India, where medical knowledge is transmitted from gods to sages and then to humans. Ayurveda therapies are based on complex herbal compounds, minerals, and metal substances. In a clinical research paper, Ayurveda was put into practice to treat a case of HE in India. The report indicates that Ayurvedic therapy, consisting on a mix of four substances (
4. Concluding remarks
Hepatic encephalopathy is a very devastating disease associated with liver failure. Besides the detrimental effects over astrocyte and neuron physiology, HE had a direct impact over the quality of life of affected patients. Recent evidence suggests that many pro-inflammatory pathways are related to these pathology and therapeutic interventions are devoted to counteract it. In this chapter, many of the molecular and pathological events related with ammonia and its effects over astrocytes cells had been addressed. Indeed, ammonia is one of the main factors contributing to the pathophysiology of HE, responsible of the astrocyte swelling along with other metabolites such as myoinositol, manganese, etc. (Figure 4). Ammonia is a toxic compound mainly produced by the bacteria in the gastrointestinal tract or by metabolism of ammonium-containing substances. In healthy subjects, urea is the final metabolite of ammonium metabolism in the liver, which is then excreted in urine. Hepatic failure is a one of the most common public concerns in western economies and is beginning to become a major problem in the next 10–15 years. Some of the most feasible and easy-to-implement therapies against liver disease is prevention. Public health policies must be strengthened regarding preventive information not only for liver but also to different metabolic-related illness. The stress and inappropriate feeding habits in general population has enormous impact not only in adults, but also in young or even in elementary-school grade childhood. One of the main tasks of public health should be informative and not only the medical practice. If we want to succeed in our struggle against different maladies affecting human beings, we must to begin to turn the sight to the basics of having a proper balanced diet and to make regular physical activity; this actions would improve and prevent any metabolic imbalance in the short to median term. The society has the future in their hands.
Figure 4.
Proposed model of astrocyte dynamic during hyperammonemic conditions. Here, the ammonia (triangle) molecules are increased in the extracellular space and they easily diffuse through the plasma membrane or by means of the NKCC1 channels (dark gray cylinders) once inside ammonia is conjugated with glutamate (
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